Human embryonic stem (hES) cells have the ability to self renew indefinitely and differentiate into virtually any cell type in the body. They offer tremendous potential for the development of new treatments for major degenerative diseases such as heart disease, Alzheimer's disease, Parkinson's, multiple sclerosis, skin aging and macular degeneration, to name a few. Recent developments in reprogramming skin cells to pluripotent cells with the same properties as embryonic stem cells have increased the availability of pluripotent stem cells for research and may ultimately provide cells for clinical applications is they can be proven safe. However, regardless of the source of pluripotent cells, there is a critical unmet need to understand how pluripotent cells differentiate in vitro to facilitate development of efficient methods of directing differentiating of pluripotent cells to desirable mature cell types for cell replacement therapy as well as for drug development. The changes in cell surface molecules that occur as pluripotent stem cells progress toward specification and commitment to various lineages are poorly understood. Mapping the surface of differentiating stem cells using traditional phage display selection methods is problematic on a mixture of heterogenous cells because the more abundant cells and receptors may mask the less common ones. We propose here to apply a novel targeted gene delivery technology combined with display library selection to directly select ligands that target lineage specific progenitors derived from hES cells (WA09). Our unique selection strategy is based on selection of the ligands using 2 gates. To be selected, the ligand-targeted vector must first internalize into the progenitor cells through a surface marker interaction. The second gate is regulated expression of a vector reporter gene in the differentiated cells that are descended from the ligand targeted progenitors. In this manner, only ligands that target progenitors of a specific differentiated cell type like cardiomyocytes as proposed here would be selected. We will first test the concept by using an EGF targeted particle to target neural progenitor cells and genetically selecting targeted particles from astrocytes. After optimizing the vector using the neural progenitor pathway, we will construct a large random peptide library and select it for cardiomyocyte targeting peptides. The selected peptides will be characterized for their ability to target and isolate cardiomyocyte progenitors. Our long term goal is to develop and commercialize targeting ligands and kits for stem cell progenitor targeting, isolation and differentiation into therapeutically useful cell-types and cells for drug screening. PUBLIC HEALTH RELEVANCE: Embryonic and other pluripotent stem cells such as recently discovered reprogrammed skin cells have the ability to self renew indefinitely and differentiate into virtually any cell type in the body. They therefore have the potential to profoundly benefit public health by providing cells for treating virtually any degenerative disease or injury such as heart disease, diabetes, MS, AD, and Parkinson's disease to name a few. It is critical to understand how pluripotent stem cells develop in the laboratory so that the right types of cells can be prepared for safe and effective regenerative treatments. The proposed project will use innovative methods to develop reagents that will facilitate stem cell research and enable the isolation, scale up and production of cells for therapy and drug development.